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Here we report the case of a cow with two ovaries that each exhibited hyperplasia but that otherwise had normal gross morphology. Both ovaries had a large number of tertiary follicles on the ovarian surface. Oocytes from one ovary were studied in more detail. The transcriptome was largely similar to other oocytes. Oocytes could undergo cleavage at a rate consistent with other oocytes and result in blastocyst-stage embryo formation after in vitro maturation and fertilization. Review of the literature from cattle and other species did not reveal reports of a similar type of spontaneous ovarian abnormality. Whole genome sequencing revealed many single nucleotide polymorphisms with predicted large effects on protein structure that could potentially be causative for the phenotype. The variant considered most likely to cause the observed alteration in ovarian function was a mutation in the glycoprotein-modifying enzyme MAN1A2. [ABSTRACT FROM AUTHOR]

Background: In beef cattle, more than 50% of the energy input to produce a unit of beef is consumed by the female that produced the calf. Development of genomic tools to identify females with high genetic merit for reproductive function could increase the profitability and sustainability of beef production. Results: Genome-wide association studies (GWAS) were performed using a single-step genomic best linear unbiased prediction approach on pregnancy outcome traits from a population of Angus–Brahman crossbred heifers. Furthermore, a validation GWAS was performed using data from another farm. Heifers were genotyped with the Bovine GGP F250 array that contains 221,077 SNPs. In the discovery population, heifers were bred in winter breeding seasons involving a single round of timed artificial insemination (AI) followed by natural mating for 3 months. Two phenotypes were analyzed: pregnancy outcome to first-service AI (PAI; n = 1,481) and pregnancy status at the end of the breeding season (PEBS; n = 1,725). The heritability was estimated as 0.149 and 0.122 for PAI and PEBS, respectively. In the PAI model, one quantitative trait locus (QTL), located between 52.3 and 52.5 Mb on BTA7, explained about 3% of the genetic variation, in a region containing a cluster of γ-protocadherin genes and SLC25A2. Other QTLs explaining between 0.5% and 1% of the genetic variation were found on BTA12 and 25. In the PEBS model, a large QTL on BTA7 was synonymous with the QTL for PAI, with minor QTLs located on BTA5, 9, 10, 11, 19, and 20. The validation population for pregnancy status at the end of the breeding season were Angus–Brahman crossbred heifers bred by natural mating. In concordance with the discovery population, the large QTL on BTA7 and QTLs on BTA10 and 12 were identified. Conclusions: In summary, QTLs and candidate SNPs identified were associated with pregnancy outcomes in beef heifers, including a large QTL associated with a group of protocadherin genes. Confirmation of these associations with larger populations could lead to the development of genomic predictions of reproductive function in beef cattle. Moreover, additional research is warranted to study the function of candidate genes associated with QTLs. [ABSTRACT FROM AUTHOR]

An embryonic circadian clock could conceivably organize cellular and developmental events temporally and in synchrony with other circadian rhythms in the mother. The hypothesis that a functional molecular clock exists in the preimplantation bovine, pig, human, and mouse embryo was tested by using publicly available RNAseq datasets to examine developmental changes in expression of the core genes responsible for the circadian clock - CLOCK, ARNTL, PER1, PER2, CRY1, and CRY2. In general, the transcript abundance of each gene decreased as development advanced to the blastocyst stage. The most notable exception was for CRY2, where transcript abundance was low and constant from the two-cell or four-cell to the blastocyst stage. Developmental patterns were generally the same for all species although there were some speciesspecific patterns such as an absence of PER1 expression in the pig, an increase in ARNTL expression at the four-cell stage in human, and an increase in expression of Clock and Per1 from the zygote to two-cell stage in the mouse. Analysis of intronic reads (indicative of embryonic transcription) for bovine embryos indicated an absence of embryonic transcription. Immunoreactive CRY1 was not detected in the bovine blastocyst. Results indicate that the preimplantation mammalian embryo lacks a functional intrinsic clock although specific components of the clock mechanism could conceivably play a role in other functions in the embryo. [ABSTRACT FROM AUTHOR]

Summary: Heat stress can have severe deleterious effects on embryo development and survival. The present study evaluated whether CSF2 can protect the developmental competence of the bovine embryo following exposure to a heat shock of 41°C at the zygote and morula stages. In the first experiment, putative zygotes and 2-cell embryos were assigned to receive either 10 ng/ml CSF2 or vehicle, and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Heat shock reduced blastocyst development for embryos treated with vehicle but not for embryos cultured with CSF2. In the second experiment, day 5 embryos (morula) were treated with CSF2 or vehicle and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Temperature treatment did not affect development to the blastocyst stage and there was no effect of CSF2 treatment or the interaction. Results indicate that CSF2 can reduce the deleterious effects of heat shock at the zygote or two-cell stage when the embryo is transcriptionally inactive. [ABSTRACT FROM AUTHOR]

Graduate education is an important aspect of the life of most academic scientists and a serious responsibility because it comes with the obligation to help students achieve their career and life goals. It can also be very fulfilling for the graduate mentor in terms of personal satisfaction and advancement of the research program. Learning to be a good major professor is an active process that depends on developing a formal framework of education and modifying that framework for each student based on past experiences and experimentation, advice from colleagues, and the individual personality of the student. Perhaps most important is for the graduate mentor to buy into the success and well-being of the student. Among the characteristics that a major professor could seek to instill in his or her students are critical and independent thinking, self-confidence, a thick skin, teamwork, laboratory skills and understanding, and the ability for hard work. Work to make science joyful by celebrating accomplishments, creating a fun environment in the lab, and stressing the societal value of science as compared to personal rewards or ambition. [ABSTRACT FROM AUTHOR]

Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase‐3 (z‐DEVD‐fmk) could improve post‐thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled‐rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1–3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 μM) and z‐DEVD‐fmk (0, 50, 100 or 200 μM), respectively. Post‐thaw survival was assessed at 24, 48 and 72 h. For experiments 4–5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post‐thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated. Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p <.05) re‐expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post‐thaw cryosurvival was not affected by the addition of either DTT or z‐DEVD‐fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p <.001) levels of intracellular ROS and fewer (p <.001) cells with DNA fragmentation. In conclusion, post‐thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate. [ABSTRACT FROM AUTHOR]

Procedures for in vitro embryo production in cattle have not been optimized. In the current experiment, we utilized a 3 × 3 factorial design to test whether the proportion of embryos becoming blastocysts in culture and the pregnancy rate after embryo transfer are affected by type of serum in the medium [no serum; 3% (v/v) KnockOut Serum Replacement (SR); 3% (v/v) fetal bovine serum (FBS)] and addition of specific embryokines [vehicle; 10 ng/mL colony stimulating factor 2 (CSF2); 100 ng/mL dickkopf related protein 1 (DKK1)] at day 5 of culture. Embryos were produced using abattoir-derived ovaries and Y-sorted semen from two Angus sires. The percent of putative zygotes and cleaved embryos becoming blastocysts was improved by SR and FBS. Pregnancy rate at day 30 was determined for 1426 Nelore recipients and calving rate for 266 recipients. In the absence of CSF2 or DKK1, pregnancy rates were lower for embryos cultured with SR or FBS. CSF2 and DKK1 reduced pregnancy rate for embryos cultured without serum but had no detrimental effect in the SR or FBS groups. Indeed, CSF2 blocked the negative effect of FBS on pregnancy rate. Data on birth weights were available for 67 bull calves. There were no effects of treatment. The sire used to produce embryos had significant and large effects on development to the blastocyst stage, pregnancy rate at day 30, calving rate and pregnancy loss between day 30 and calving. Results indicate that (1) SR and FBS can improve embryonic development in vitro while also compromising competence of embryos to survive after transfer, (2) actions of CSF2 and DKK1 depend upon other characteristics of the embryo production system, and (3) sire can have a large effect on embryonic development before and after transfer. [ABSTRACT FROM AUTHOR]

Exponential proliferation of trophoblast stem cells (TSC) is crucial in Ruminantia to maximize numerical access to caruncles, the restricted uterine sites that permit implantation. When translating systems biology of the undifferentiated bovine trophectoderm, we uncovered that inhibition of RhoA/Rock promoted self-renewing proliferation and substantially increased blastocyst size. Analysis of transcripts suppressed by Rock inhibition revealed transforming growth factor ß1 (TGFß1) as a primary upstream effector. TGFß1 treatment induced changes consistent with differentiation in bTSCs, a response that could be replicated by induced expression of the bovine ROCK2 transgene. Rocki could partially antagonize TGFß1 effects, and TGFß receptor inhibition promoted proliferation identical to Rocki, indicating an all-encompassing upstream regulation. Morphological differentiation included formation of binucleate cells and infrequent multinucleate syncytia, features we also localize in the in vivo bovine placenta. Collectively, we demonstrate a central role for TGFß1, RhoA and Rock in inducing bTSC differentiation, attenuation of which is sufficient to sustain self-renewal and proliferation linked to blastocyst size and preimplantation development. Unraveling these mechanisms augments evolutionary/comparative physiology of the trophoblast cell lineage and placental development in eutherians. [ABSTRACT FROM AUTHOR]

In cattle, starting 4-5 days after estrus, preimplantation embryonic development occurs in the confinement of the uterine lumen. Cells in the endometrial epithelial layer control the molecular traffic to and from the lumen and, thereby determine luminal composition. Starting early postestrus, endometrial function is regulated by sex steroids, but the effects of progesterone on luminal cells transcription have not been measured in vivo. The first objective was to determine the extent to which progesterone controls transcription in luminal epithelial cells 4 days (D4) after estrus. The second objective was to discover luminal transcripts that predict pregnancy outcomes when the effect of progesterone is controlled. Endometrial luminal epithelial cells were collected from embryo transfer recipients on D4 using a cytological brush and their transcriptome was determined by RNASeq. Pregnancy by embryo transfer was measured on D30 (25 pregnant and 18 nonpregnant). Progesterone concentration on D4 was associated positively (n = 182) and negatively (n = 58) with gene expression. Progesterone-modulated transcription indicated an increase in oxidative phosphorylation, biosynthetic activity, and proliferation of epithelial cells. When these effects of progesterone were controlled, different genes affected positively (n = 22) and negatively (n = 292) odds of pregnancy. These set of genes indicated that a receptive uterine environment was characterized by the inhibition of phosphoinositide signaling and innate immune system responses. A panel of 25 genes predicted the pregnancy outcome with sensitivity and specificity ranging from 64%-96% and 44%-83%, respectively. In conclusion, in the early diestrus, both progesterone-dependent and progesterone-independent mechanisms regulate luminal epithelial transcription associated with pregnancy outcomes in cattle. [ABSTRACT FROM AUTHOR]

This study aimed to identify the impact of supplementing rumen-protected methionine (RPM) during the periconceptional period on beef cows in their progeny. Evidence suggests that changes in maternal nutrition in the periconceptional period have effects during the postnatal growth phase in cattle. We hypothesized that feeding RPM during the periconceptional period would program bovine gestation to enhance progeny growth. A total of 114 beef cows were fed a roughage-based diet and randomized to receive corn gluten supplemented with 15 g of RPM (Smartamine M, Adisseo) or not (CON) from d -7 to +7 relative to artificial insemination (AI) with female sex-sorted semen. Pregnancy diagnoses were conducted using transrectal ultrason-ography 30 and 60 d after AI. Female calves (n = 34) remained with their dams until weaning. A month after weaning, calves were weighed every 14 d during an 8-mo period, and wither height (WH), body length (BL), and heart girth (HG) were measured. Dry matter intake (DMI), average daily gain (ADG), and residual feed intake (RFI) were measured during a 56-d performance test. At the end of the performance test, the 12th rib back fat (BF), ribeye area (REA), intramuscular fat (IMF), and rump fat (RF) were determined via ultrasound. A treatment-by-day interaction tendency (P = 0.10) was observed in HG, having larger HG in the RPM treatment from d 336-adj of age (CON = 154.52 ± 1.46 cm; RPM = 158.36 ± 1.34 cm). During the performance test, animals in the RPM treatment were heavier (CON = 437.25 ± 2.46 kg; RPM = 442.79 ± 2.34 kg; P = 0.01) at the end of the test. However, no treatment difference (P > 0.05) was observed in DMI (CON = 12.06 ± 0.19 kg/d; RPM = 12.14 ± 0.18 kg/d), ADG (CON = 1.50 ± 0.03 kg; RPM = 1.55 ± 0.03 kg) and RFI (CON = -0.12 ± 0.15; RPM = 0.11 ± 0.14). No treatment difference (P > 0.05) was observed in BF, REA, and IMF. However, RF was greater in the control treatment (CON = 1.16 ± 0.07 cm; RPM = 0.91 ± 0.06 cm), and a tendency of having a thicker BF in control was observed (Table 1). In conclusion, supplementing RPM enhanced final body weight during the performance test; furthermore, a treatment-by-day interaction tendency was observed in HG. On the contrary, CON progeny had more RF and a tendency to have a thicker BF. [ABSTRACT FROM AUTHOR]

In beef cattle, 40% of cows that display estrus do not remain pregnant after embryo transfer (ET). Estrus is a physiological behavior influenced by the concentration of sex-steroids. The use of accelerometer devices for estrus detection is a common practice in dairy operations. It remains unclear if this same artificial intelligence software can accurately measure reproductive behavior in beef females. We hypothesized that estrous variables detected by an accelerometer are associated with ovarian, hormonal, pregnancy outcome, and fertility-related variables measured in the cow. Fifty Bos indicus-influenced, cyclic, primiparous cows were fitted with an accelerometer collar (SCR Engineers, Israel) and a breeding indicator (Estrotectâ) to detect estrus after a synchronization protocol in five subsequent experimental replicates (n = 50*5 = 250). Only cows that showed estrus (d 0) remained in the study. The following variables were used from the accelerometer software: Activity Peak, Rumination Nadir, Estrus Duration, and Heat Index, calculated by proprietary algorithms. Dominant follicle (DF) diameter and plasma concentration of estradiol were measured on d -2 (day of progesterone device removal) and on d 0. On d -1 and d 4, endometrial thickness and luminal fluid accumulation were measured by ultrasonography. On d 7, two embryos were transferred/cow. Pregnancies were diagnosed on d 45 and terminated. After 30 d, a new replicate started. Cows included in at least two replicates that remained pregnant 100%, 99-50%, or less than 50% were classified as fertile, sub fertile, and infertile, respectively. Estrus display was recorded for 166 cows (66%). Estrus detection by accelerometer and Estrotect had excellent agreement rate (kappa coefficient 0.76). Cows detected in estrus only by Estrotect had 66% less pregnancy/ET than cows in estrus by both (9/21 vs. 97/145; P = 0.01). The DF diameter on d 0 was not associated with accelerometer variables. Cows with greater and moderate Activity Peak had a greater growth rate of the DF from d -2 to d 0 than lower Activity Peak cows (interaction day*Activity Peak; P = 0.06). Estradiol on d -1 was positively associated with the Activity Peak (P < 0.0001). Cows with lower Activity Peak had a thicker endometrium on both d -1 and d 4 (P=0.02), and greater uterine luminal fluid score on d -1 (P=0.03). Fertility status was not associated with any accelerometer variables. In conclusion, the Activity Peak is associated positively with estradiol concentration and DF growth, however no association was found with pregnancy outcome and fertility. Therefore, the main advantage of accelerometers in beef ranches is the ability to detect estrus at a similar accuracy as breeding indicators, using less labor. Future enhancements in hardware and software to allow prediction of pregnancy outcomes are expected to increase reproductive efficiency in cow-calf operations. [ABSTRACT FROM AUTHOR]

In beef cattle, for a single pregnancy opportunity, only 60% of cows will remain pregnant. It is possible that uterine dysfunction prevents adequate embryo signaling and subsequent placentation. Our aim was to compare the abundance of molecular markers associated with pregnancy retention among embryo recipients classified for fertility. The hypothesis was that expression of interferon-stimulated genes (ISG15 and RSAD2) in lymphocytes and concentration of pregnancy associated glycoproteins (PAGs) in serum increase according to cow fertility. Cyclic, Bos Indicus-influenced, primiparous cows (n = 50) were submitted to five consecutive rounds of estrus synchronization, embryo transfer (ET), pregnancy termination and recovery. The ET was conducted 7 days after estrus (D7). Each cow received two embryos per transfer from a randomly assigned bull pair (BP) containing one embryo from each bull A, B or C (BP1 = A+B; BP2 = A+C; BP3 = B+C). Pregnancy diagnostic was conducted on D32 and D45 using B-mode ultra-sonography. Blood samples were collected on D20 to quantify expression of ISG15 and RSAD2 in peripheral blood mononuclear cells and on D25, D32, and D39 to quantify serum concentration of PAGs. Based on D45 pregnancy outcome (pregnant or open), cows were classified as Fertile (Fert; n = 14), Sub-fertile (SF; n = 16) or Infertile (IF; n = 15) if they were pregnant 3, 2, or 1 out of the 5 experimental rounds, respectively. Five cows that never sustained a pregnancy until D45 were excluded. The overall prevalence of twin-pregnancies was 7.8% (n = 13/166). Expression of ISG15 was increased in Fert compared to IF cows (P = 0.01; 0.0196 ± 0.0042 vs 0.0164 ± 0.0052, respectively). There was no effect of fertility classification on the expression of RSAD2. Serum concentrations of PAGs increased over time (P < 0.0001), regardless of fertility classification (day x fertility classification; P > 0.4). There was an effect of BP on the probability of pregnancy on D32 and D45 (P < 0.05). For D32, BP2 (84%) was 3 times more likely to maintain a pregnancy than BP1 (52%), while BP3 (74%) was not significantly different. On D45, BP2 (80%) was 2.8 times more likely to remain pregnant than BP1 (50%), and BP3 (62%) was not different than either BP1 or BP2. The interaction bull pair by fertility classification on the probability of pregnancy on D45 approached significance (P = 0.1). While pregnancy to BP2 was similar across all fertility classifications (80%), pregnancy to BP1 decreased dramatically as classifications changed from Fert to IF (88% vs. 14%, respectively). In conclusion, IF cows were less able to respond to IFN-t, likely because of a reduced secretion of IFN-t from the elongating conceptus. Also, IF cows are more susceptible to the genetics of the embryo than FERT cows. [ABSTRACT FROM AUTHOR]

Alterations in the environment of the preimplantation embryo can affect competence to establish pregnancy and phenotype of resultant calves. In this study, the bovine embryo produced in vitro was used to evaluate postnatal programming actions of the embryokine colony-stimulating factor 2 (CSF2) and serum, which is a common additive of culture media. Oocytes were collected by ovum pick up from Brahman donors and fertilized with semen from Brahman bulls. Embryos were randomly assigned to one of the three treatments: vehicle, CSF2 10 ng/mL, or 1% (v/v) serum. Treatments were added to the culture medium from day 5 to 7 after fertilization. Blastocysts were harvested on day 7 and transferred into crossbred recipients. Postnatal body growth and Longissimus dorsi muscle characteristics of the resultant calves were measured. The percent of cleaved embryos becoming blastocysts was increased by serum and, to a lesser extent, CSF2. Treatment did not affect survival after embryo transfer but gestation length was shortest for pregnancies established with serum-treated embryos. Treatment did not significantly affect postnatal body weight or growth. At 3 mo of age, CSF2 calves had lower fat content in the Longissimus dorsi muscle and less subcutaneous fat over the muscle than vehicle calves. There was a tendency for cross-sectional area of the muscle to be smaller for serum calves than vehicle calves. Results confirm the importance of the preimplantation period as a window to modulate postnatal phenotype of resultant calves. In particular, CSF2 exerted actions during the preimplantation period to program characteristics of accumulation of intramuscular and subcutaneous fat of resultant calves. The use of a low serum concentration in culture medium from day 5 to 7 of development can increase the yield of transferrable embryos without causing serious negative consequences for the offspring. [ABSTRACT FROM AUTHOR]

Typically, bovine embryos are transferred into recipient females about day 7 after estrus or anticipated ovulation, when the embryo has reached the blastocyst stage of development. All the biological and technical causes for failure of a female to produce a blastocyst 7 d after natural or artificial insemination (AI) are avoided when a blastocyst-stage embryo is transferred into the female. It is reasonable to expect, therefore, that pregnancy success would be higher for embryo transfer (ET) recipients than for inseminated females. This expectation is not usually met unless the recipient is exposed to heat stress or is classified as a repeat-breeder female. Rather, pregnancy success is generally similar for ET and AI. The implication is that either one or more of the technical aspects of ET have not yet been optimized or that underlying female fertility that causes an embryo to die before day 7 also causes it to die later in pregnancy. Improvements in pregnancy success after ET will depend upon making a better embryo, improving uterine receptivity, and forging new tools for production and transfer of embryos. Key to accelerating progress in improving pregnancy rates will be the identification of phenotypes or phenomes that allow the prediction of embryo competence for survival and maternal capacity to support embryonic development. [ABSTRACT FROM AUTHOR]

Tunnel ventilation is an increasingly popular approach to mitigate the effects of heat stress on dairy cattle. Tunnel-ventilation barns use a bank of high-power fans to move air horizontally from one end of the barn to the other at cow level. The overall objective of the present experiments was to determine whether tunnel ventilation is superior to housing with fans and sprinklers with respect to rectal temperature during heat stress and seasonal variation in milk yield. In the first study, rectal temperatures were measured for 1097 lactating Holstein cows in six freestall barns with fans and sprinklers and 575 lactating Holsteins in four tunnel-ventilated freestall barns at a time point between 14:00 and 16:00 h during the months of June to August in Florida, USA. Rectal temperatures were lower for cows in tunnel-ventilation barns than sprinkler-and-fan barns when the tunnel-ventilation barns were built de novo but not when the tunnel-ventilation barns were produced by retrofitting a sprinkler-and-fan barn (interaction, P = 0.0129). In the second study, average daily milk yield in the first 90 days in milk was examined for 8470 lactating Holsteins housed in three sprinkler-and-fan barns and two tunnel-ventilation barns. Milk production for cows calving in cool weather (October to March) was greater (P < 0.0001) than for cows calving in hot weather (April to September). The seasonal reduction in milk yield was less for cows (P = 0.037) in tunnel-ventilation barns (3.5% decrease) than for cows in sprinkler-and-fan barns (5.8% decrease). With this difference in impact of heat stress, it was estimated at a dairy farm could invest up to a $332 more per cow space in a tunnel-ventilated barn than in a sprinkler-and-fan barn. It was concluded that housing cows in tunnel-ventilation barns can reduce the impact of heat stress on body temperature regulation and milk yield. [ABSTRACT FROM AUTHOR]

Colony-stimulating factor 3 (CSF3), also known as granulocyte colony-stimulating factor, is used to reduce the incidence of mastitis in cattle. Here, we tested whether recombinant bovine CSF3 at 1, 10, or 100 ng/mL acts on the bovine oocyte during maturation or on the developing embryo to modify competence for development and characteristics of the resultant blastocyst. For experiment 1, oocytes were matured with or without CSF3. The resultant embryos were cultured in a serumfree medium for 7.5 d. There was no effect of CSF3 on cleavage or on development to the blastocyst stage except that 100 ng/mL reduced the percent of putative zygotes and cleaved embryos becoming blastocysts. Expression of transcripts for 93 genes in blastocysts was evaluated by RT-PCR using the Fluidigm platform. Transcript abundance was affected by one or more concentrations of CSF3 for four genes only (CYP11A1, NOTCH2, RAC1, and YAP1). For experiment 2, cumulusoocyte complexes (COC) were fertilized with either X- or Y-sorted semen. Putative zygotes were cultured in medium containing CSF3 treatments added at the beginning of culture. There was no effect of CSF3, sex, or the interaction on the percent of putative zygotes that cleaved or on the percent of putative zygotes or cleaved embryos becoming a blastocyst. For experiment 3, CSF3 was added from day 4 to 7.5 of development. There was no effect of CSF3 on development to the blastocyst stage. Transcript abundance of 10 genes was increased by 100 ng/mL CSF3, including markers of epiblast (NANOG, SOX2), hypoblast (ALPL, FN1, KDM2B, and PDGFRA), epiblast and hypoblast (HNF4A) and trophectoderm (TJAP1). Results are indicative that concentrations of CSF3 higher than typical after therapeutic administration can reduce oocyte competence and act on the embryo to affect characteristics of the blastocyst. [ABSTRACT FROM AUTHOR]

The mammalian embryo displays sexual dimorphism in the preimplantation period. Moreover, competence of the embryo to develop is dependent on the sire from which the embryo is derived and can be modified by embryokines produced by the endometrium such as colony stimulating factor 2 (CSF2). The preimplantation period is characterized by large changes in epigenetic modifications of DNA and histones. It is possible, therefore, that effects of sex, sire, and embryo regulatory molecules are mediated by changes in epigenetic modifications. Here it was tested whether global levels of two histone modifications in the trophectoderm of the bovine blastocyst were affected by sex, sire, and CSF2. It was found that amounts of immunolabeled H3K27me3 were greater (P = 0.030) for male embryos than female embryos. Additionally, labeling for H3K27me3 and H3K18ac depended upon the bull from which embryos were derived. Although CSF2 reduced the proportion of embryos developing to the blastocyst, there was no effect of CSF2 on labeling for H3K27me3 or H3K18ac. Results indicate that the blastocyst trophoctoderm can be modified epigenetically by embryo sex and paternal inheritance through alterations in histone epigenetic marks. [ABSTRACT FROM AUTHOR]

Thermal stress in hot humid conditions limits cattle production. The objectives for this study were to estimate genetic parameters for hair characteristics and core body temperature under low and high temperature humidity index (THI) conditions. Hair samples were collected and measured for length and diameter. Core body temperature was measured as vaginal temperature every 15 min over a 5-d period using an iButton temperature measuring device implanted in a blank CIDR in 336 heifers from the University of Florida multibreed herd (ranging from 100% Angus to 100% Brahman). Restricted maximum likelihood procedures were used to estimate heritabilities from multiple bivariate animal models using the WOMBAT program. Estimates of heritability for hair diameter, undercoat length, topcoat length, body temperature under low THI conditions, and body temperature under high THI conditions were 0.50, 0.67, 0.42, 0.32, and 0.26, respectively. The genetic parameters estimated in this study indicate a large, exploitable genetic variance which can be selected upon to improve tolerance in cattle. Breed effects for differing compositions of Brahman and Angus were also estimated. As Brahman breed composition increased by 25% undercoat length, topcoat length, body temperature under low THI conditions, and body temperature under high THI conditions decreased by 1.32 mm, 2.94 mm, 0.11 °C, and 0.14 °C, respectively. Under both low and high THI conditions, cattle with 25% Brahman breed composition or greater maintained a significantly lower body temperature than the 100% Angus breed group. The incorporation of Brahman germplasm is recommended for herds that often experience heat stress conditions in order to increase resilience to heat stress. [ABSTRACT FROM AUTHOR]

Summary: Heat shock may disrupt oocyte function by increasing the generation of reactive oxygen species (ROS). We evaluated the capacity of the antioxidant melatonin to protect oocytes using two models of oxidative stress – heat shock and the pro-oxidant menadione. Bovine cumulus–oocyte complexes (COC) were exposed in the presence or absence of 1 µM melatonin to the following treatments during maturation: 38.5°C, 41°C and 38.5°C+5 µM menadione. In the first experiment, COC were matured for 3 h with 5 µM CellROX® and analyzed by epifluorescence microscopy to quantify production of ROS. The intensity of ROS was greater for oocytes exposed to heat shock and menadione than for control oocytes. Melatonin reduced ROS intensity for heat-shocked oocytes and oocytes exposed to menadione, but not for control oocytes. In the second experiment, COC were matured for 22 h. After maturation, oocytes were fertilized and the embryos cultured for 7.5 days. The proportion of oocytes that cleaved after fertilization was lower for oocytes exposed to heat shock and menadione than for control oocytes. Melatonin increased cleavage for heat-shocked oocytes and oocytes exposed to menadione, but not for control oocytes. Melatonin tended to increase the developmental competence of embryos from heat-shocked oocytes but not for embryos from oocytes exposed to menadione or from control oocytes. In conclusion, melatonin reduced production of ROS of maturing oocytes and protected oocytes from deleterious effects of both stresses on competence of the oocyte to cleave after coincubation with sperm. These results suggest that excessive production of ROS compromises oocyte function. [ABSTRACT FROM AUTHOR]

The objective was to determine whether pregnancy success after embryo transfer (ET) during heat stress in multi-service Holstein cows depends upon characteristics of the embryo or recipient. Female embryos produced in vitro were cultured with either 0.0 (control) or 1.8 mM choline chloride and transferred fresh. Fresh embryos of undetermined breed and frozen Holstein embryos were used when experimental embryos were insufficient. Embryos were transferred 8 d after the last GnRH injection of an ovulation synchronization program. Embryo type [frozen vs. fresh, choline vs. control, unknown breed vs. (control + choline)] and characteristics of recipients (average of 190 d in milk at transfer) were evaluated. Pregnancy per ET was lower for cows receiving frozen embryos (7.0%; 3/43) than for cows receiving fresh embryos (26.7%; 32/120) but there were no differences between various types of fresh embryo. Pregnancy per ET was lower for cows diagnosed with metritis in the early postpartum period (7.1%; 2/28) than for cows without metritis (24.4%; 33/135). In conclusion, the use of frozen/thawed embryos produced in vitro and recipients which had metritis in the early postpartum period reduced the success of ET in multiple-service Holstein cows. [ABSTRACT FROM AUTHOR]

Heat stress is a cause of major economic losses to cattle producers, especially in tropical and subtropical environments. The objectives of this study were to assess the phenotypic variability in core body temperature and sweating rate and to evaluate the effect of coat type, temperament, and BW on core body temperature and sweating rate in Brangus heifers. During August and September of 2016, 725 Brangus heifers were evaluated on pasture in four separate groups (n = 200, 189, 197, and 139). Environmental measurements of dry bulb temperature (Tdb) and relative humidity (RH) were measured every 15 min during the entire time of data collection and the temperature-humidity index (THI) was used to quantify heat-stress potential. Coat score, sweating rate, chute score, exit score, and live weight were recorded as the animals passed through the chute. Vaginal temperature was recorded every 5 min for five consecutive days. There was significant variation in vaginal temperature between heifers in the same environmental conditions (s² u = 0.049), suggesting opportunities for selective improvements. A repeatability of 0.47 and 0.44 was estimated for sweating rate and vaginal temperature, respectively, suggesting that one measurement would be able to adequately describe the sweating capacity or ability to control the body temperature of an individual. Vaginal temperature increased as THI increased, with approximately 1 h lag time in the animal's response. Vaginal temperature (-0.047 °C, P = 0.015) and sweating rate were lower (-5.49 ± 2.12 g/(m2·h), P < 0.01) for heifers that demonstrated a calmer behavior in the chute. Animals with shorter, smoother hair coats had significantly lower vaginal temperatures when compared to animals with longer hair coats (P < 0.01). Also, heavier heifers in this study maintained lower (P < 0.0001) vaginal temperature than the lighter heifers. Our results showed that hair coat, temperament, and weight influenced vaginal temperature regulation. [ABSTRACT FROM AUTHOR]

The objective was to evaluate the influence of varying amounts of Brahman genetics on body temperature under pasture conditions during hot weather. Vaginal temperatures were measured at 5-min intervals for 3 to 5 d on four occasions during August and September from a total of 190 pregnant cows that were either Angus, 2/8 Brahman (remainder Angus), Brangus (3/8 Brahman), 4/8 Brahman, 6/8 Brahman or Brahman. Vaginal temperature was higher for the first two replicates than for the second two replicates. In the first two replicates, average vaginal temperature did not differ between genetic groups, but average vaginal temperature from 1500 to 1900 h was lower for Brahman than other groups. In the second two replicates, average vaginal temperature was lower for cows that were 4/8 or higher Brahman than for cows that were 2/8 Brahman or Angus. Average vaginal temperature from 1500 to 1900 h was lower for cows that were 4/8 or higher Brahman than for cows that were 2/8 Brahman or Angus. In addition, Brahman cows had lower vaginal temperatures than cows that were 4/8 Brahman or 3/8 Brahman (i.e., Brangus). In one replicate, a tracking device was used to map cow location. At 1200 to 1300 h, cows that were 6/8 Brahman or Brahman had fewer observations near the tree line (i.e., in shade) than cows that were 4/8 Brahman or less. At 1500 to 1600 h, cows that were 4/8 or higher Brahman experienced fewer observations near the tree line than cows that contained a lower fraction of Brahman genetics. In summary, a minimum of 4/8 Brahman genetics was required to increase the ability to regulate body temperature and at least 6/8 Brahman when heat stress was severe. It is likely, therefore, that using Brahman genetics to optimize adaptation to thermal stress under conditions of severe heat stress requires a preponderance of Brahman genes. [ABSTRACT FROM AUTHOR]

Evolutionary adaptations are occasionally convergent solutions to the same problem. A mutation contributing to a heat tolerance adaptation in Senepol cattle, a New World breed of mostly European descent, results in the distinct phenotype known as slick, where an animal has shorter hair and lower follicle density across its coat than wild type animals. The causal variant, located in the 11th exon of prolactin receptor, produces a frameshift that results in a truncated protein. However, this mutation does not explain all cases of slick coats found in criollo breeds. Here, we obtained genome sequences from slick cattle of a geographically distinct criollo breed, namely Limonero, whose ancestors were originally brought to the Americas by the Spanish. These data were used to identify new causal alleles in the 11th exon of the prolactin receptor, two of which also encode shortened proteins that remove a highly conserved tyrosine residue. These new mutations explained almost 90% of investigated cases of animals that had slick coats, but which also did not carry the Senepol slick allele. These results demonstrate convergent evolution at the molecular level in a trait important to the adaptation of an animal to its environment. [ABSTRACT FROM AUTHOR]

A major unresolved issue is how the uterus influences infertility and subfertility in cattle. Serial embryo transfer was previously used to classify heifers as high-fertile (HF), subfertile (SF), or infertile (IF). To assess pregnancy loss, two in vivo-produced embryos were transferred into HF, SF, and IF heifers on day 7, and pregnancy outcome was assessed on day 17. Pregnancy rate was substantially higher in HF (71%) and SF (90%) than IF (20%) heifers. Elongating conceptuses were about twofold longer in HF than SF heifers. Transcriptional profiling detected relatively few differences in the endometrium of nonpregnant HF, SF and IF heifers. In contrast, there was a substantial difference in the transcriptome response of the endometrium to pregnancy between HF and SF heifers. Considerable deficiencies in pregnancy-dependent biological pathways associated with extracellular matrix structure and organization as well as cell adhesion were found in the endometrium of SF animals. Distinct gene expression differences were also observed in conceptuses from HF and SF animals, with many of the genes decreased in SF conceptuses known to be embryonic lethal in mice due to defects in embryo and or placental development. Analyses of biological pathways, key players and ligand-receptor interactions based on transcriptome data divulged substantial evidence for dysregulation of conceptus-endometrial interactions in SF animals. These results support the ideas that the uterus impacts conceptus survival and programs conceptus development and ripple effects of dysregulated conceptus-endometrial interactions elicit loss of the postelongation conceptus in SF cattle during the implantation period of pregnancy. [ABSTRACT FROM AUTHOR]

Infertility and subfertility negatively impact the economics and reproductive performance of cattle. Of note, significant pregnancy loss occurs in cattle during the first month of pregnancy, yet little is known about the genetic loci influencing pregnancy success and loss in cattle. To identify quantitative trait loci (QTL) with large effects associated with early pregnancy loss, Angus crossbred heifers were classified based on day 28 pregnancy outcomes to serial embryo transfer. A genome wide association analysis (GWAA) was conducted comparing 30 high fertility heifers with 100% success in establishing pregnancy to 55 subfertile heifers with 25% or less success. A gene set enrichment analysis SNP (GSEA-SNP) was performed to identify gene sets and leading edge genes influencing pregnancy loss. The GWAA identified 22 QTL (p < 1 x 10−5), and GSEA-SNP identified 9 gene sets (normalized enrichment score > 3.0) with 253 leading edge genes. Network analysis identified TNF (tumor necrosis factor), estrogen, and TP53 (tumor protein 53) as the top of 671 upstream regulators (p < 0.001), whereas the SOX2 (SRY [sex determining region Y]-box 2) and OCT4 (octamer-binding transcription factor 4) complex was the top master regulator out of 773 master regulators associated with fertility (p < 0.001). Identification of QTL and genes in pathways that improve early pregnancy success provides critical information for genomic selection to increase fertility in cattle. The identified genes and regulators also provide insight into the complex biological mechanisms underlying pregnancy establishment in cattle. [ABSTRACT FROM AUTHOR]

The first two differentiation events in the embryo result in three cell types - epiblast, trophectoderm (TE) and hypoblast. The purpose here was to identify molecular markers for each cell type in the bovine and evaluate the differences in gene expression among individual cells of each lineage. The cDNA from 67 individual cells of dissociated blastocysts was used to determine transcript abundance for 93 genes implicated as cell lineage markers in other species or potentially involved in developmental processes. Clustering analysis indicated that the cells belonged to two major populations (clades A and B) with two subpopulations of clade A and four of clade B. Use of lineage-specific markers from other species indicated that the two subpopulations of clade A represented epiblast and hypoblast respectively while the four subpopulations of clade B were TE. Among the genes upregulated in epiblast were AJAP1, DNMT3A, FGF4, H2AFZ, KDM2B, NANOG, POU5F1, SAV1 and SLIT2. Genes overexpressed in hypoblast included ALPL, FGFR2, FN1, GATA6, GJA1, HDAC1, MBNL3, PDGFRA and SOX17, while genes overexpressed in all four TE populations were ACTA2, CDX2, CYP11A1, GATA2, GATA3, IFNT, KRT8, RAC1 and SFN. The subpopulations of TE varied among each other for multiple genes including the prototypical TE marker IFNT. New markers for each cell type in the bovine blastocyst were identified. Results also indicate heterogeneity in gene expression among TE cells. Further studies are needed to confirm whether subpopulations of TE cells represent different stages in the development of a committed TE phenotype. [ABSTRACT FROM AUTHOR]

The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. In the present study we tested four growth factors expressed in the endometrium for their ability to modify the development of the bovine embryo to the blastocyst stage and alter the expression of genes found to be upregulated (bone morphogenetic protein 15 (BMP15) and keratin 8, type II (KRT8)) or downregulated (NADH dehydrogenase 1 (ND1) and S100 calcium binding protein A10 (S100A10)) in embryos competent to develop to term. Zygotes were treated at Day 5 with 0.01, 0.1 or 1.0nM growth factor. The highest concentration of activin A increased the percentage of putative zygotes that developed to the blastocyst stage. Connective tissue growth factor (CTGF) increased the number of cells in the inner cell mass (ICM), decreased the trophectoderm : ICM ratio and increased blastocyst expression of KRT8 and ND1. The lowest concentration of hepatocyte growth factor (HGF) reduced the percentage of putative zygotes becoming blastocysts. Teratocarcinoma-derived growth factor 1 increased total cell number at 0.01nM and expression of S100A10 at 1.0 nM, but otherwise had no effects. Results confirm the prodevelopmental actions of activin A and indicate that CTGF may also function as an embryokine by regulating the number of ICM cells in the blastocyst and altering gene expression. Low concentrations of HGF were inhibitory to development. [ABSTRACT FROM AUTHOR]

The goal was to gain understanding of how 12 genes containing SNP previously related to embryo competence to become a blastocyst (BRINP3, C1QB, HSPA1L, IRF9, MON1B, PARM1, PCCB, PMM2, SLC18A2, TBC1D24, TTLL3 and WBP1) participate in embryonic development. Gene expression was evaluated in matured oocytes and embryos. BRINP3 and C1QB were not detected at any stage. For most other genes, transcript abundance declined as the embryo developed to the blastocyst stage. Exceptions were for PARM1 and WBP1, where steady-state mRNA increased at the 9–16 cell stage. The SNP in WBP1 caused large differences in the predicted three-dimensional structure of the protein while the SNP in PARM1 caused smaller changes. The mutation in WBP1 causes an amino acid substitution located close to a P-P-X-Y motif involved in protein-protein interactions. Moreover, the observation that the reference allele varies between mammalian species indicates that the locus has not been conserved during mammalian evolution. Knockdown of mRNA for WBP1 decreased the percent of putative zygotes becoming blastocysts and reduced the number of trophectoderm cells and immunoreactive CDX2 in the resulting blastocysts. WBP1 is an important gene for embryonic development in the cow. Further research to identify how the SNP in WBP1 affects processes leading to differentiation of the embryo into TE and ICM lineages is warranted. [ABSTRACT FROM AUTHOR]

The bovine was used to examine the potential for WNT signaling to affect the preimplantation embryo. Expression of seven key genes involved in canonical WNT signaling declined to a nadir at the morula or blastocyst stage. Expression of 80 genes associated with WNT signaling in the morula and inner cell mass (ICM) and trophectoderm (TE) of the blastocyst was also evaluated. Many genes associated with WNT signaling were characterized by low transcript abundance. Seven genes were different between ICM and TE, and all of them were overexpressed in TE as compared to ICM, including WNT6, FZD1, FZD7, LRP6, PORCN, APC and SFRP1. Immunoreactive CTNNB1 was localized primarily to the plasma membrane at all stages examined from the 2-cell to blastocyst stages of development. Strikingly, neither CTNNB1 nor non-phospho (i.e., active) CTNNB1 was observed in the nucleus of blastomeres at any stage of development even after the addition of WNT activators to culture. In contrast, CTNNB1 associated with the plasma membrane was increased by activators of WNT signaling. The planar cell polarity pathway (PCP) could be activated in the embryo as indicated by an experiment demonstrating an increase in phospho-JNK in the nucleus of blastocysts treated with the non-canonical WNT11. Furthermore, WNT11 improved development to the blastocyst stage. In conclusion, canonical WNT signaling is attenuated in the preimplantation bovine embryo but WNT can activate the PCP component JNK. Thus, regulation of embryonic development by WNT is likely to involve activation of pathways independent of nuclear actions of CTNNB1. [ABSTRACT FROM AUTHOR]

• Embryo transfer can improve genetic selection, fertility and postnatal phenotypes. • Overcoming inefficiencies in the system will make the practice more widespread. • One goal is to increase the number of oocytes from genetically elite females. • Another is to increase the efficiency of generating a live calf from each oocyte. • It may be possible to program development to optimize calf postnatal phenotype. The embryo produced by in vitro oocyte maturation, fertilization, and embryonic development is an important resource for genetic improvement and has the potential to improve female fertility and to be programmed to produce offspring with superior ability for health and production. The cultured embryo is also an important component of several realized and potential technologies such as gene editing, somatic cell nuclear cloning, stem cell technologies and gamete generation in vitro. Full realization of the opportunities afforded by the in vitro -produced embryo will require overcoming some technical obstacles to cost-effective implementation of an embryo transfer program. Among the research goals for improving the penetration of embryo transfer in the cattle industry are development of methods to increase the supply of oocytes from genetically elite females, enhance the proportion of oocytes that become transferrable embryos, improve the fraction of embryos that establish pregnancy after transfer, reduce pregnancy wastage after pregnancy diagnosis, and identify culture conditions to optimize postnatal phenotype. [ABSTRACT FROM AUTHOR]

We tested whether gene expression of the bovine morula is modified by CSF2 in a sex-dependent manner and if sex determines the effect of CSF2 on competence of embryos to become blastocysts. Embryos were produced in vitro using X- or Y-sorted semen and treated at Day 5 of culture with 10 ng/mL bovine CSF2 or control. In experiment 1, morulae were collected at Day 6 and biological replicates (n = 8) were evaluated for transcript abundance of 90 genes by RT-qPCR using the Fluidigm Delta Gene assay. Expression of more than one-third (33 of 90) of genes examined was affected by sex. The effect of CSF2 on gene expression was modified by sex (P < 0.05) for five genes (DDX3Y/DDX3X-like, NANOG, MYF6, POU5F1 and RIPK3) and tended (P < 0.10) to be modified by sex for five other genes (DAPK1, HOXA5, PPP2R3A, PTEN and TNFSF8). In experiment 2, embryos were treated at Day 5 with control or CSF2 and blastocysts were collected at Day 7 for immunolabeling to determine the number of inner cell mass (ICM) and trophectoderm (TE) cells. CSF2 increased the percent of putative zygotes that became blastocysts for females, but did not affect the development of males. There was no effect of CSF2 or interaction of CSF2 with sex on the total number of blastomeres in blastocysts or in the number of inner cell mass or trophectoderm cells. In conclusion, CSF2 exerted divergent responses on gene expression and development of female and male embryos. These results are evidence of sexually dimorphic responses of the preimplantation embryo to this embryokine. [ABSTRACT FROM AUTHOR]

Background: Colony stimulating factor 2 can have multiple effects on the function of the preimplantation embryo that include increased potential to develop to the blastocyst stage, reduced apoptosis, and enhanced ability of inner cell mass (ICM) to remain pluripotent after culture. The objective of the current experiment was to identify genes regulated by CSF2 in the ICM and trophectoderm (TE) of the bovine blastocyst with the goal of identifying possible molecular pathways by which CSF2 increases developmental competence for survival. Embryos were produced in vitro and cultured from Day 6 to 8 in serum-free medium containing 10 ng/ml recombinant bovine CSF2 or vehicle. Blastocysts were harvested at Day 8 and ICM separated from TE by magnetic-activated cell sorting. RNA was purified and used to prepare amplified cDNA, which was then subjected to high-throughput sequencing using the SOLiD 4.0 system. Three pools of amplified cDNA were analyzed per treatment%. Results: The number of genes whose expression was regulated by CSF2, using P < 0.05 and >1.5-fold difference as cut-offs, was 945 in the ICM (242 upregulated by CSF2 and 703 downregulated) and 886 in the TE (401 upregulated by CSF2 and 485 downregulated). Only 49 genes were regulated in a similar manner by CSF2 in both cell types. The three significant annotation clusters in which genes regulated by ICM were overrepresented were related to membrane signaling. Genes downregulated by CSF2 in ICM were overrepresented in several pathways including those for ERK and AKT signaling. The only significant annotation cluster containing an overrepresentation of genes regulated by CSF2 in TE was for secreted or extracellular proteins. In addition, genes downregulated in TE were overrepresented in TGFß and Nanog pathways%. Conclusions: Differentiation of the blastocyst is such that, by Day 8 after fertilization, the ICM and TE respond differently to CSF2. Analysis of the genes regulated by CSF2 in ICM and TE are suggestive that CSF2 reinforces developmental fate and function of both cell lineages. [ABSTRACT FROM AUTHOR]

Sex affects function of the developing mammalian embryo as early as the preimplantation period. There were two goals of the current objective. The first was to determine the degree and nature of differences in gene expression between female and male embryos in the cow at the morula stage of development. The second objective was to determine whether DKK1, a molecule known to alter differentiation of the blastocyst, would affect gene expression differently for female and male morulae. In Experiment 1, female and male embryos were treated with DKK1 at Day 5 after insemination. Morulae were harvested 24 h after treatment, pooled in groups of 20 for microarray analysis and RNA subjected to analysis of gene expression by microarray hybridization. There were 662 differentially expressed genes between females and males and 128 of these genes had a fold change ≥ 1.5 between the two sexes. Of the genes upregulated in females, 49.5% were located in the X chromosome. Functional analysis predicted that cell survival was greater in female embryos. Experiment 2 involved a similar design except that transcripts for 12 genes previously reported to be affected by sex, DKK1 or the interaction were quantified by quantitative polymerase chain reaction. Expression of all genes tested that were affected by sex in experiment 1 was affected in a similar manner in Experiment 2. In contrast, effects of DKK1 on gene expression were largely not repeatable in Experiment 2. The exception was for the Hippo signaling gene AMOT, which was inhibited by DKK1. In Experiment 3, embryos produced by fertilization with unsorted sperm were treated with DKK1 at Day 5 and abundance of transcripts for CDX2, GATA6, and NANOG determined at Days 5, 6 and 7 after insemination. There was no effect of DKK1 on expression of any of the three genes. In conclusion, female and male bovine embryos have a different pattern of gene expression as early as the morula stage, and this is due to a large extent to expression of genes in the X chromosomes in females. Differential gene expression between female and male embryos is likely the basis for increased resistance to cell death signals in female embryos and disparity in responses of female and male embryos to changes in the maternal environment. [ABSTRACT FROM AUTHOR]

Socrates defends justice against the attack mounted by Thrasymachus in Plato's Republic, but it isn't easy to say what the defense consists of. A careful reading shows that Thrasymachus himself is deeply though unselfconsciously attached to justice. Thrasymachus admires skillful artisans and thinks that devoting oneself to one's art makes one a good man, worthy of good things. He feels that the skillful and unjust deserve to get the better of the artless and weak, which is to say he unselfconsciously believes in a form of distributive justice. Moreover, his surprising attachment to justice resembles and casts light on the attachment to justice that most people feel. [ABSTRACT FROM AUTHOR]

Clusters of imprinted genes are often controlled by an imprinting center that is necessary for allele-specific gene expression and to reprogram parent-of-origin information between generations. An imprinted domain at 15q11--q13 is responsible for both Angelman syndrome (AS) and Prader--Willi syndrome (PWS), two clinically distinct neurodevelopmental disorders. Angelman syndrome arises from the lack of maternal contribution from the locus, whereas Prader--Willi syndrome results from the absence of paternally expressed genes. In some rare cases of PWS and AS, small deletions may lead to incorrect parent-of-origin allele identity. DNA sequences common to these deletions define a bipartite imprinting center for the AS--PWS locus. The PWS--smallest region of deletion overlap (SRO) element of the imprinting center activates expression of genes from the paternal allele. The AS--SRO element generates maternal allele identity by epigenetically inactivating the PWS--SRO in oocytes so that paternal genes are silenced on the future maternal allele. Here we have investigated functional activities of the AS--SRO, the element necessary for maternal allele identity. We find that, in humans, the AS--SRO is an oocyte-specific promoter that generates transcripts that transit the PWS--SRO. Similar upstream promoters were detected in bovine oocytes. This result is consistent with a model in which imprinting centers become DNA methylated and acquire maternal allele identity in oocytes in response to transiting transcription. [ABSTRACT FROM AUTHOR]

Previous evidence has suggested that fertilization and preimplantation embryonic development are sensitive periods that can be influenced by changes in maternal nutrition. We hypothesized that feeding rumen-protected methionine (RP-Met) during the periconceptional period will program bovine gestation in a manner that enhances fetal and postnatal growth. A total of 114 cows were fed a roughage-based diet and randomized to receive corn gluten supplemented with 15 g of RP-Met (RP-Met; Smartamine, Adisseo) or not (Con) from d -7 to +7 relative to artificial insemination (AI) with sex-sorted semen. Estrus synchronization was conducted using the 7-day CoSynch + CIDR protocol. Pregnancy diagnosis, embryo, and fetal morphology were conducted using transrectal ultrasonography 30, and 60 days after AI. After calving, 40 calves (Cont = 19; RP-Met = 21) were considered for birth weight (BW) analysis. Female calves (n=34) were weighed at 2 months of age. Pregnancy per AI at day 30 (Con = 50%, RP-Met = 55%) and day 60 (Con = 50%; RP-Met = 53.4%) were not different between groups (P ≥ 0.05). At 30 days of pregnancy, embryonic vesicle diameter (Con=11.0 ± 0.7 mm; RP-Met =11.6 ± 0.7 mm; P < 0.05) and circumference (Con=0.88 ± 0.09 cm2; RP-Met =0.99 ± 0.09 cm² P < 0.05) were larger in the RP-Met group but embryo length and abdominal cavity were not different between groups. No difference was observed in fetal morphology at d 60. A treatment by sex interaction was observed (P =0.04). Male calves (n=6) from cows fed RP-Met showed greater BW than those from Con group (Con=31.9 ± 2.3 kg; RP-Met =41.4 ± 2.3 kg) but no difference was observed in females (n=34). There was no difference in BW at 2 months of age between groups. In conclusion, using RP-Met increased embryonic vesicle diameter and circumference, and BW in males. [ABSTRACT FROM AUTHOR]

Reproduction is among the physiological functions in mammals most susceptible to disruption by hyperthermia. Many of the effects of heat stress on function of the oocyte and embryo involve direct effects of elevated temperature (i.e. heat shock) on cellular function. Mammals limit the effects of heat shock by tightly regulating body temperature. This ability is genetically controlled: lines of domestic animals have been developed with superior ability to regulate body temperature during heat stress. Through experimentation in cattle, it is also evident that there is genetic variation in the resistance of cells to the deleterious effects of elevated temperature. Several breeds that were developed in hot climates, including Bos indicus (Brahman, Gir, Nelore and Sahiwal) and Bos taurus (Romosinuano and Senepol) are more resistant to the effects of elevated temperature on cellular function than breeds that evolved in cooler climates (Angus, Holstein and Jersey). Genetic differences are expressed in the preimplantation embryo by Day 4-5 of development (after embryonic genome activation). It is not clear whether genetic differences are expressed in cells in which transcription is repressed (oocytes >100 µm in diameter or embryos at stages before embryonic genome activation). The molecular basis for cellular thermotolerance has also not been established, although there is some suggestion for involvement of heat shock protein 90 and the insulin-like growth factor 1 system. Given the availability of genomic tools for genetic selection, identification of genes controlling cellular resistance to elevated temperature could be followed by progress in selection for those genes within the populations in which they exist. It could also be possible to introduce genes from thermotolerant breeds into thermally sensitive breeds. The ability to edit the genome makes it possible to design new genes that confer protection of cells from stresses like heat shock. [ABSTRACT FROM AUTHOR]

Successful embryonic development is dependent on factors secreted by the reproductive tract. Dickkopf-1 (DKK1), an antagonist of the wingless-related mouse mammary tumor virus (WNT) signaling pathway, is one endometrial secretory protein potentially involved in maternal-embryo communication. The purpose of this study was to investigate the roles of DKK1 in embryo cell fate decisions and competence to establish pregnancy. Using in vitro-produced bovine embryos, we demonstrate that exposure of embryos to DKK1 during the period of morula to blastocyst transition (between d 5 and 8 of development) promotes the first 2 cell fate decisions leading to increased differentiation of cells toward the trophectoderm and hypoblast lineages compared with that for control embryos treated with vehicle. Moreover, treatment of embryos with DKK1 or colony-stimulating factor 2 (CSF2; an endometrial cytokine known to improve embryo development and pregnancy establishment) between d 5 and 7 of development improves embryo survival after transfer to recipients. Pregnancy success at d 32 of gestation was 27% for cows receiving control embryos treated with vehicle, 41% for cows receiving embryos treated with DKK1, and 39% for cows receiving embryos treated with CSF2. These novel findings represent the first evidence of a role for maternally derived WNT regulators during this period and could lead to improvements in assisted reproductive technologies. [ABSTRACT FROM AUTHOR]

• The SLICK1 mutation in bovine PRLR confers cattle with a short hair coat and increased ability for thermoregulation. • Consequences for other physiological functions are not well understood. • It was found that animals with the mutation have slight alterations in hepatic gene expression. • Thus, inheritance of the SLICK1 allele can exert functional changes beyond those related to hair growth. • The mutation acts as a dominant allele because there were few differences in body temperature and liver gene expression between animals with one or two copies of the gene. The SLICK1 mutation in bovine PRLR (c.1382del; rs517047387) is a deletion mutation resulting in a protein with a truncated intracellular domain. Cattle carrying at least one allele have a phenotype characterized by a short hair coat (slick phenotype) and increased resistance to heat stress. Given the pleiotropic nature of prolactin, the mutation may affect other physiological characteristics. The liver is one organ that could potentially be affected because of the expression of PRLR. The mutation is a dominant allele, and heterozygous animals have a similar hair coat to that of animals homozygous for the mutation. Present objectives were to determine whether inheritance of the SLICK1 mutation affects liver gene expression and if animals homozygous for the SLICK1 allele differ from heterozygotes in liver gene expression and regulation of body temperature during heat stress. In one experiment, rectal and ruminal temperatures were less for Holstein heifers that were heterozygous for the SLICK1 allele compared with wildtype heifers. There were 71 differentially expressed genes in liver, with 13 upregulated and 58 downregulated in SLICK1 heterozygotes. Among the ontologies characteristic of differentially expressed genes were those related to immune function and fatty acid and amino acid metabolism. In a prospective cohort study conducted with adult Senepol cattle, body temperature and hepatic gene expression were compared between animals heterozygous or homozygous for the SLICK1 mutation. There were no differences in ruminal temperatures between genotypes, rectal temperature was higher in animals homozygous for the SLICK1 mutation, and there was only one gene in liver that was differentially expressed. It was concluded that inheritance of the SLICK1 allele can exert functional changes beyond those related to hair growth although changes in liver gene expression were not extensive. Results are also consistent with the SLICK1 allele being dominant because there were few differences in phenotype between animals inheriting one or two copies of the allele. [ABSTRACT FROM AUTHOR]

Background: Identification of single nucleotide polymorphisms (SNPs) for specific genes involved in reproduction might improve reliability of genomic estimates for these low-heritability traits. Semen from 550 Holstein bulls of high (⩾ 1.7; n = 288) or low (⩽ -2; n = 262) daughter pregnancy rate (DPR) was genotyped for 434 candidate SNPs using the Sequenom MassARRAY® system. Three types of SNPs were evaluated: SNPs previously reported to be associated with reproductive traits or physically close to genetic markers for reproduction, SNPs in genes that are well known to be involved in reproductive processes, and SNPs in genes that are differentially expressed between physiological conditions in a variety of tissues associated in reproductive function. Eleven reproduction and production traits were analyzed. Results: A total of 40 SNPs were associated (P < 0.05) with DPR. Among these were genes involved in the endocrine system, cell signaling, immune function and inhibition of apoptosis. A total of 10 genes were regulated by estradiol. In addition, 22 SNPs were associated with heifer conception rate, 33 with cow conception rate, 36 with productive life, 34 with net merit, 23 with milk yield, 19 with fat yield, 13 with fat percent, 19 with protein yield, 22 with protein percent, and 13 with somatic cell score. The allele substitution effect for SNPs associated with heifer conception rate, cow conception rate, productive life and net merit were in the same direction as for DPR. Allele substitution effects for several SNPs associated with production traits were in the opposite direction as DPR. Nonetheless, there were 29 SNPs associated with DPR that were not negatively associated with production traits. Conclusion: SNPs in a total of 40 genes associated with DPR were identified as well as SNPs for other traits. It might be feasible to include these SNPs into genomic tests of reproduction and other traits. The genes associated with DPR are likely to be important for understanding the physiology of reproduction. Given the large number of SNPs associated with DPR that were not negatively associated with production traits, it should be possible to select for DPR without compromising production. [ABSTRACT FROM AUTHOR]

Heat stress compromises production, fertility, and health of dairy cattle. One mitigation strategy is to select individuals that are genetically resistant to heat stress. Most of the negative effects of heat stress on animal performance are a consequence of either physiological adaptations to regulate body temperature or adverse consequences of failure to regulate body temperature. Thus, selection for regulation of body temperature during heat stress could increase thermotolerance. The objective was to perform a genome-wide association study (GWAS) for rectal temperature (RT) during heat stress in lactating Holstein cows and identify SNPs associated with genes that have large effects on RT. Records on afternoon RT where the temperature-humidity index was ≥78.2 were obtained from 4,447 cows sired by 220 bulls, resulting in 1,440 useable genotypes from the Illumina BovineSNP50 BeadChip with 39,759 SNP. For GWAS, 2, 3, 4, 5, and 10 adjacent SNP were averaged to identify consensus genomic regions associated with RT. The largest proportion of SNP variance (0.07 to 0.44%) was explained by markers flanking the region between 28,877,547 and 28,907,154 bp on Bos taurus autosome (BTA) 24. That region is flanked by U1 (28,822,883 to 28,823,043) and NCAD (28,992,666 to 29,241,119). In addition, the SNP at 58,500,249 bp on BTA 16 explained 0.08% and 0.11% of the SNP variance for 2- and 3-SNP analyses, respectively. That contig includes SNORA19, RFWD2 and SCARNA3. Other SNPs associated with RT were located on BTA 16 (close to CEP170 and PLD5), BTA 5 (near SLCO1C1 and PDE3A), BTA 4 (near KBTBD2 and LSM5), and BTA 26 (located in GOT1, a gene implicated in protection from cellular stress). In conclusion, there are QTL for RT in heat-stressed dairy cattle. These SNPs could prove useful in genetic selection and for identification of genes involved in physiological responses to heat stress. [ABSTRACT FROM AUTHOR]

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6–8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6–8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation. [ABSTRACT FROM AUTHOR]

Background: While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3′ tag digital gene expression (3′DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance. Results: Using 3′DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT. Conclusions: Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage. [ABSTRACT FROM AUTHOR]